Terrestrial Ecology, National Environmental Research Institute, Silkeborg, Denmark. *Author for correspondence (

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1 4 The Journal of Experimental Biology 9, Pulished y The Company of Biologists 6 doi:.14/je.484 Seasonal aquisition of hill tolerane and restruturing of memrane glyerophospholipids in an overwintering inset: triggering y low temperature, desiation and diapause progression A. Tomala 1,, M. Tollarová 1,, J. Overgaard 3, P. Simek 1 and V. Kostál 1,, 1 Biology Centre AS CR, Institute of Entomology, Branisovská 31, 37 Ceské Budejovie, Czeh Repuli, Faulty of Biologial Sienes, University of South Bohemia, Ceské Budejovie, Czeh Repuli and 3 Department of Terrestrial Eology, National Environmental Researh Institute, Silkeorg, Denmark Author for orrespondene ( kostal@entu.as.z) Aepted August 6 Adults of the inset Pyrrhooris apterus aquire hill tolerane through the proess of autumnal alimatization. Field and laoratory experiments were onduted to separate the triggering effets of low temperatures, desiation and diapause progression on the physiologial harateristis related to hill tolerane with emphasis on the restruturing of glyerophospholipid (GPL) omposition. Changes in relative proportions of major moleular speies of glyerophosphoethanolamines (GPEtns) and glyerophosphoholines (GPChols) in thorai musle and fat ody tissues were followed using HPLC oupled to eletrospray ionisation mass spetrometry. The inrease in relative proportion of 1- palmitoyl--linoleyl-sn-gpetn at the expense of 1,- dilinoleyl-sn-gpchol was the most prominent feature of the omplex hange oserved in oth tissues during autumnal alimatization in the field. The relative Summary proportion of total GPEtns inreased, while the proportion of total GPChols dereased. The relative proportion of unsaturated fatty ayls slightly dereased. A similar restruturing response was seen during alimatization in the field and old alimation in the laoratory. By ontrast, the GPL hanges related to desiation and diapause progression were relatively small, differed qualitatively from the old-alimation response, and were aompanied with no inrease of hill tolerane. Other features of autumnal alimatization, i.e. depression of superooling apaity and aumulation of polyhydri alohols, were also triggered solely y low temperatures. Key words: memrane phospholipids, temperature, alimatization, old alimation, desiation, diapause, old tolerane, etotherm, Inseta, Heteroptera. Introdution Poikilotherm organisms, from ateria, through plants to animals, show apaity for remodelling of the lipid omposition of their iologial memranes in response to hanging amient temperature (Sinensky, 1974; Steponkus, 199; Cossins, 1994; Hazel, 199; Hazel, 1997; Wallis and Browse, ). Cold alimation harateristially leads to one or a omination of the following adjustments as summarized y Hazel (Hazel, 1989; Hazel, 199): (a) inreased proportion of unsaturated fatty aids; () restruturing of glyerophospholipid (GPL) moleular speies; () elevated relative proportions of glyerophosphoethanolamines (GPEtns) to glyerophosphoholines (GPChols); (d) redued proportions of plasmalogens ompared to diayl GPLs; (e) adjustment of holesterol ontent. Suh hanges has een postulated to prevent perturation of memrane struture and funtion when the ody temperature hanges. Diret geneti proof for the role of inreased unsaturation in enhaning survival at dereasing amient temperatures has een otained in yanoateria and plants (Browse et al., 1994; Murata, 1994; Allakhverdiev et al., 1999; Inaa et al., 3). Also in insets, alimatory responses to old has een shown to involve restruturing of GPL omposition in iologial memranes (Bennet et al., 1997; Ohtsu et al., 1998; Kostál and Simek, 1998; Hodková et al., 1999; Hodková et al., ; Bayley et al., 1; Holmstrup et al., ; Slahta et al., a; Kostál et al., 3; Overgaard et al., ). Dereasing amient temperature is most often onsidered to e the triggering fator for alimatory hanges in GPL omposition. In insets, however, the triggering fators and transdution asades for GPL ompositional hanges have not een analyzed in detail. Based on earlier studies, at least three different triggering mehanisms might e involved: amient temperature, hydration state of the organism

2 Cold alimation of memranes 43 (mediated via osmolarity/onentration of speifi solutes) and endogenous fators suh as developmental hormones. In the field situation, all three fators may operate in parallel, whih makes it diffiult to distinguish etween their effets. Changes in temperature intervene oth daily and seasonally. Naturally ourring diurnal variations in temperature were suffiient to indue the noturnal inrease of old tolerane in adults of Drosophila melanogaster through the proess of rapid old hardening (Lee et al., 1987) and this was aompanied y small ut signifiant hanges in the GPL omposition (Overgaard et al., ). Many insets that overwinter in temperate and polar haitats enhane their old hardiness during the alimatization proess, whih starts upon entering to diapause and ontinues during autumnal derease of amient temperatures (Denlinger, 1991; Slahta et al., ). Changes in GPL omposition were oserved oth in the field and in the laoratory studies during suh winter old hardening (Bennet et al., 1997; Hodková et al., 1999; Hodková et al., ; Kostál et al., 3). Partial dehydration often represents an inseparale part of the omplex alimatory hange related to old alimation (Ring and Danks, 1994; Blok, 1996; Danks, ; Williams et al., 4). It raises the question of whether the GPL hanges oserved during old alimation may e triggered y hanges in hydration state of the organism. Indeed, typial memrane alterations resulting in a higher degree of unsaturation were indued y mild desiation stress applied to the ollemolan Folsomia andida (Bayley et al., 1; Holmstrup et al., ). Many insets hange their hormonal milieu seasonally, in response to speifi token stimuli suh as photoperiod, and enter diapause (Denlinger, 198; Tauer et al., 1986; Danks, 1987; Kostál, 6). Similar remodelling of GPL omposition was oserved in response to old exposure and to transition from diret development to diapause in two insets, adult P. apterus (Hodková et al., ), and larvae of the fly Chymomyza ostata (Kostál et al., 3). The main ojetive of this study was to desrie the nature and triggering fators of seasonal hanges in GPL omposition in the ug Pyrrhooris apterus L. (Inseta: Heteroptera). The hanges oserved during autumnal alimatization of wild ugs were ompared with the alimation hanges indued under ontrolled laoratory onditions, where three potential triggering/modulating fators were examined separately: low temperatures, desiation and progression of diapause. Survival at su-zero temperatures, and several other physiologial parameters related to it, were measured in parallel and linked to the hanges in GPL omposition Materials and methods Insets, alimations In the present study, wild and laoratory-reared insets were ompared. Wild adults were olleted in Ceské Budejovie (Czeh Repuli) on two oasions during, on 3rd Otoer and 7th Deemer. All insets were in reprodutive diapause and they passed through the proess of winter old hardening etween Otoer and Deemer (Hodek, 1968; Kostál and Simek, ). Fig. 1A shows the air temperatures as reorded y the weather station of the Czeh Institute of Hydrometeorology in Ceské Budejovie. The inset samples were transported to the laoratory ( min) without hanging their temperature and were then proessed immediately. Laoratory-reared insets ame from a ulture that originated from adults olleted in Ceské Budejovie during spring. F emryos and nymphs were reared under onditions induing reprodutive diapause in the adults, i.e. onstant temperature of C and a short-day photoperiod of 1 h:1 h, L:D (Hodek, 1968). Dry seeds of the linden tree A Field Field daily max. Field daily min. B Laoratory Adult moult state progression Temperature ( C) Coldalimation 3 Otoer 7 Deemer Otoer Novemer Deemer Days Fig. 1. Course of air temperatures in the field (A) and the temperature protool used for laoratory alimation (B) of Pyrrhooris apterus. The adult insets were sampled on 3 Otoer and 7 Deemer in the field. Laoratory adults were maintained under short days onditions (1 h:1 h, L:D). Their physiologial state was onsidered as the initial state when they reahed age of 4 weeks. Three different treatments were then applied: progression of diapause, desiation, and old alimation (see text for detailed desriptions).

3 44 A. Tomala and others (Tilia parviflora Ehrh.) and water were provided ad liitum. Adult F insets were kept at a onstant C, with a short-day photoperiod and with aess to food and water, for 4 weeks. Suh adults were onsidered to e in deep reprodutive diapause and their physiologial state was onsidered as the initial state. Three different treatments were then applied (depited in Fig. 1B). (1) Progression of diapause: the insets were kept under onstant onditions (aess to food and water) for another 8 weeks. () : the insets were kept at C, with a short-day photoperiod, and denied aess to liquid water and food for 14 days. Relative humidity of the air flutuated etween 4 6%. Suh a treatment results in approximately % mortality (Tollarová and V.K., unpulished data), and in the present study only survivors were taken for analyses. (3) Cold alimation: the insets were exposed to gradually dereasing temperatures: C (day)/ C (night) during the first week, followed y C/ C and C/ C during the seond and third weeks, respetively (all 3 weeks in short days), followed y weeks at onstant C and ontinuous darkness. Suh an alimation protool simulates the natural drop of temperatures during autumn (see Fig. 1A) and the ugs ahieve higher hill tolerane than if onstant temperatures (in ontrast to thermoperiodi regime) are used for alimation (Kostál et al., 1). Physiologial parameters The fresh mass (FM) was measured in five males and five females (individually) using a Sartorius alane with sensitivity of.1 mg. Dry mass (DM) was measured after drying the speimens at 6 C for 3 days. Hydration (in mg H O mg 1 DM) was alulated from the gravimetri data. Haemolymph samples for osmolality measurements were olleted from insets (five males, five females) y utting off one of the antennae and allowing it to leed into a alirated apillary tue. Osmolalities were measured in -nl droplets using the Clifton Nanoliter Osmometer (Clifton Tehnial Physis, USA). The onentrations of the two most aundant winter polyols (riitol and soritol) that typially aumulate in diapausing old-alimated P. apterus adults (Kostál and Simek, ; Kostál et al., 1), were measured in 7% ethanol extrats of whole ody samples (five males, five females, taken individually). The extration, derivatization and analytial proedures (gas hromatography oupled to mass spetrometry) were the same as desried y Kostál and Simek (Kostál and Simek, 199). All hemials used for analysis were purhased from Sigma-Aldrih Co. (St Louis, MO, USA). The temperature of rystallization of ody fluids (superooling point, SCP) was measured in individual insets (16 males, 16 females) at a ooling rate of.3 min 1 using the method desried (Nedved et al., 199). Chill tolerane was assessed y exposing groups of insets ( males, females) to a temperature of C (maximum, 14. C; minimum,.3 C) for various periods (1, 4, 7,, 13, or 8 days). The apaity for oordinated rawling at C, days after the exposure, was used as a riterion for survival. Survival rate was desried using the parameter LT, whih gives the time of exposure that is susequently survived y % of the population sample. The parameter Lt was alulated from nonlinear logisti regression with the sigmoid urve as desried (Kostál et al., 1). Analysis of glyerophospholipids Thorai musle and fat ody tissues were rapidly (3 min) disseted from the adults. Tissues from three individuals were pooled to otain one sample, and eah sample was repliated six times (three times with males, three times with females). Total lipids were extrated in ie-old hloroform:methanol (:1) solution using the method of Folh et al. (Folh et al., 197) slightly modified aording to Kostál and Simek (Kostál and Simek, 1998). After the extration, the solvents were evaporated under a stream of nitrogen, and lipids were stored at 8 C until analysis. High performane liquid hromatography (HPLC) omined with eletrospray ionisation mass spetrometry (ESI-MS) (Han and Gross, 199; Hsu and Turk, ; Brooks et al., ; Wang et al., ) was performed on a quadrupole ion LCQ mass spetrometer (Thermo, San Jose, CA, USA) oupled to a Rheos ternary HPLC system (Flux, Basel, Switzerland), equipped with a FAMOS autosampler and Thermos thermostat (oth LC Pakings-Dionex, Amsterdam, The Netherlands). The stored dry samples were dissolved in 1 ml of methanol and l aliquots were injeted into a mm i.d. 3. m Synergi Polar HPLC olumn (Phenomenex, Torrane, CA, USA). The moile phase was omposed of (A) mmol l 1 ammonium aetate (NH 4 A) in methanol, (B): mmol l 1 NH 4 A in water and (C) isopropanol. A linear gradient of A:B:C hanging from 9:: to 7::3 within 14 min was used with a flow rate of 3 l min 1. The olumn temperature was maintained at 3 C. The mass spetrometer was operated either in positive or negative ion detetion modes at +4 kv or 3.6 kv, respetively. Capillary temperature was 4 C; nitrogen was used as oth the sheath and the auxiliary gas. For MS and MS3 fragmentations, ion isolation windows were and Da, respetively. Maximum ion injetion time was ms, ollision energies were 3% (MS) or 3% (MS3), mass range of 6 8 Da was sanned eah. s. All hemials used for extration and analysis were purhased from Sigma-Aldrih Co. Data proessing As there were no signifiant differenes etween males and females, the data sets for oth sexes were pooled. Owing to the omplexity of instrument response to partiular GPL moleular speies, whih is dependent not only on features of the hemial struture ut also on experimental onditions, results of GPL analysis were preferaly expressed in relative values, i.e. relative molar proportion of eah lipid speies from the total of %. Our analysis was foused on major GPEtn and GPChol moleular speies, whih were present at or aove 1%. It is known that GPEtns plus GPChols represent more than 8% of all glyerophospholipids in P. apterus tissues (Hodková et al.,

4 Cold alimation of memranes ). The ratio of total GPEtns to total GPChols (GPEtn/GPChol) was alulated for eah tissue/treatment and the relative proportions of individual fatty ayls were alulated ased on GPL data. Unsaturation ratio (UFA/SFA) was alulated from the proportional omposition of fatty ayls. Unpaired two-tailed t-tests (parametri) were used to ompare means of two variales with normal Gaussian distriution (F-tests were used to verify equality of varianes). Mann Whitney U-tests (non-parametri) were used to ompare the medians of SCP variales (with skewed distriution of data). To investigate if there were any general trends in the hanges of GPL omposition aused y different treatments, all data were analysed together using a multivariate statistial analysis, Prinipal omponent analysis (PCA; Sima-P software, Umetris, Umea, Sweden). Using this method, the data are represented in K-dimensional spae (where K is equal to the numer of variales) and redued to a few prinipal omponents (two prinipal omponents, PC1 and PC, in this study) that desrie the maximum variation within the data. The prinipal omponents are then displayed as a set of sores, whih highlight lustering. Differenes etween the mean sores for different treatments along the axes PC1 and PC were tested using one-way ANOVA followed y a post-ho Student Newman Keuls test (SNK). Results Changes in hill tolerane and related parameters Wild adults of P. apterus olleted in the field during Otoer and Deemer had similar dry masses (Tale 1). Although their hydration dereased from Otoer to Deemer, this hange was statistially insignifiant (P=.61; t-test). Osmolality of haemolymph, however, inreased almost threefold (from 36 to 94 mosmol kg 1 ) and high amounts of Tale 1. Physiologial parameters in the field-olleted adults of Pyrrhooris apterus Value in Value in Parameter Otoer Deemer Dry mass (mg) 18.1± ±3.7 Hydration (mg water mg 1 DM) 1.78± ±.3 Haemolymph osmolality 36±1 94± (mosmol kg 1 ) Con. of riitol ( g mg 1 DM) trae 6.96±8.14 Con. of soritol ( g mg 1 DM) trae 3.8±.88 Superooling point ( C) (.8,.) ( 16.4,.3) Lt at C (days) <1 18.9±3.4 DM, dry mass. Eah value represents either a mean ± s.d. or, in the ase of superooling points, median (%, 7% perentile); (see text for N). The differenes etween Otoer and Deemer means or medians were statistially analyzed using unpaired two-tailed t-tests or twotailed Mann Whitney U-tests, respetively (P<.; P<.1; P<.1). winter polyols aumulated: 7. and 3.8 g mg 1 DM of riitol and soritol, respetively. The temperature of rystallization of ody fluids (SCP) dereased from 11. in Otoer to 19. C in Deemer and the hill tolerane (measured as a Lt at C) inreased from <1 to 18.9 days. There were no signifiant differenes in the measured physiologial parameters etween the laoratory-reared insets during their initial state and those olleted in the field in Otoer (ompare Tales 1 and ; P>. for all parameters, t-tests or U-test). When laoratory adults were sujeted to various treatments, their dry mass did not hange signifiantly (Tale ). Hydration remained stale during the progression of diapause at C, ut it signifiantly dereased during the desiation and old-alimation treatments. Desiating onditions aused the loss of 1% of initial water ontent and an approximate twofold inrease in haemolymph osmolality. Cold alimation was aompanied y the loss of 34% of initial water ontent and more than a threefold inrease in osmolality. No winter polyols aumulated during the progression of diapause. Small amounts of polyols (slightly aove the analytial thresholds:. or 1. g of riitol or soritol, respetively, in the sample) were found in desiated insets and high amounts of polyols, i.e..9 and 11.4 g mg 1 DM of riitol and soritol, respetively, were found in the oldalimated speimens. Superooling apaity and hill tolerane at C remained relatively low during diapause progression and desiation while old alimation aused the SCP to derease from 13.9 to 18.9 C and the Lt at C to inrease from <1 to 14.6 days. Glyerophospholipid omposition Gradient HPLC effiiently separated GPLs from other lipid lasses, partiularly from triaylglyerols, whih were dominant in the sample extrats. In this way, highly reproduile full-san positive ESI mass spetra of the GPChol and GPEtn frations were otained with minimal outward peak overlapping and ion suppression. As shown in Fig., [M+H] + ions dominated in the full-san ESI spetra of GPEtn and GPChol frations. Twelve to 13 major GPEtn and GPChol moleular speies were onsistently deteted in the tissue samples of P. apterus (Figs, 3). Fig. 3 shows a typial HPLC hromatogram (upper trae), and extrated hromatograms of prinipal GPEtn and GPChol speies. The identity of eah major ion was verified y fragmenting the ion and studying the MS and MS3 spetra ontaining preferential losses of headgroups and fatty ayls from the sn-1 or sn- positions of the glyerol akone. All GPL speies were omposed of only five different fatty ayls (FAs): palmitoyl (16:), heptadeanoyl (17:), stearoyl (18:), oleyl (18:1) and linoleyl (18:). The GPL ompositions were qualitatively similar in the wild (Otoer) and laoratory-reared (initial state) insets in oth tissues (ompare Fig. 4A and Fig. A with Fig. 6A and Fig. 7A, respetively). There were, however, quantitative differenes at the level of individual GPL speies. The single largest differene was found in the proportion of - GPChol in the musle tissue, whih was 8.% in the wild

5 46 A. Tomala and others Tale. Physiologial parameters in the laoratory-reared adults of Pyrrhooris apterus, and the effets of diapause progression, desiation and old alimation state After diapause After desiation After old Parameter (day ) progression ( months) (14 days) alimation ( months) Dry mass (mg) 1.± ± ± ±6. Hydration (mg water mg 1 DM) 1.7±.1 1.6±.33.8±. 1.13±.14 Haemolymph osmolality (mosmol kg 1 ) 34±7 373±4 7±11 118±37 Con. of riitol ( g mg 1 DM) trae trae.34±.7.94±6.4 Con. of soritol ( g mg 1 DM) trae trae.11± ±. Superooling point ( C) 13.9 ( 1., 14.8) 1.9 ( 6.,.) 11.7 ( ) 18.9 ( 14.9, 19.6) Lt at C (days) <1 <1 <1 14.6±4.1 DM, dry mass. Eah value represents either a mean ± s.d. or, in the ase of superooling points, median (%, 7% perentile); (see text for N). The differenes etween the initial state and the treatments were statistially analyzed using unpaired two-tailed t-tests or two-tailed Mann Whitney U-tests, respetively (P<.; P<.1; P<.1). Value insets and only 19.4% in the laoratory-reared insets (P<.1, t-test). Wild speimens showed similar GPEtn/GPChol ratios in oth tissues (musle, 1.±.3; fat ody,.9±.). By ontrast, laoratory-reared insets had a ratio of 1.±.3 in the musles and.±.1 in the fat ody. Seasonal hange in glyerophospholipid omposition The omplex hange in GPL omposition during seasonal alimatization in the field is shown in Figs 4 and. Two most notieale alterations were: (1) the inrease of 16:/18:- GPEtn y 6.6 or.3% (musle or fat, respetively), and () the Relative aundane A GPEtns B GPChols 716,3 74, , 74, , , 717, ,4 73,4 786, 746,3 79, 766,4 78, 8, 718,3 738,4 731,4 86, 76,4 787, 767,4 7,3 78,4 76, 77, 7, 78,3 747, 719, 7, 73,4 73,4 763,4 761,6 768,4 777,1 89, 7,6 78, 713,1 749,3 77, 744,3 749,1 77,6 773, 788,4 794, 73,3 761, 76, 77,7 776,3 798,1 83,3 8,8 818, m/z 744,3 Fig.. Eletrospray ionisation mass spetra showing moleular speies omposition of P. apeterus thorai musle tissue. Positive full mass spetra of (A) glyerophosphoethanolamine (GPEtn) and (B) glyerophosphoholine (GPChol) frations are depited separately. Identities of all major [M+H] + moleular ions (solid ovals) were verified y MS and MS3 fragmentations and are shown in Fig. 3. Note that eah major ion ours as a pair of masses differing y 1 amu ( 13 C isotopi ontriution). All speies formed the adduts with sodium [M+Na] + in relatively low and onstant proportions (roken ovals) Ion mass (amu) 78, 78, m/z

6 Cold alimation of memranes 47 derease of -GPChol y 4.7% (oth tissues). The GPEtn/GPChol ratios signifiantly inreased from Otoer to Deemer: to 1.4±. in the musles (P=.11, t-test), and to 1.±. in the fat ody (P=.93, t-test). FA omposition hanged relatively little during autumnal alimatization (Tale 3). Statistially signifiant inreases in the proportion of palmitoyl (16:) were registered in oth tissues, and the proportions of linoleyl (18:) tended to derease. Suh hanges resulted in a slight, ut statistially signifiant, derease in UFA/SFA ratio in oth tissues. Changes in glyerophospholipid omposition during laoratory alimations The diapause progression and desiation treatments were aompanied y relatively small hanges in GPL omposition of the musle (Fig. 6B,C) and fat ody (Fig. 7B,C). The relative proportions of GPEtn/GPChol remained unhanged (P>., t- test): musle after diapause progression, 1.4±.3; musle after desiation, 1.6±.4; fat ody after diapause progression,.6±.1; and fat ody after desiation.6±.1. Relatively small hanges were oserved in FA omposition, and the UFA/SFA ratios did not hange signifiantly (Tale 4). The old-alimation treatment resulted in onsiderale restruturing of GPL omposition. The general patterns of hanges were similar in oth tissues (ompare Fig. 6D with Fig. 7D). The proportion of 16:/18:-GPEtn inreased y 8.1% and 3.3% in the musle and fat ody tissue, respetively; 18:/18:-GPEtn was the seond most inreased speies (y.9% and 3.%). Also, -GPChol dereased y 8.8% and 6.%, and 16:/18:-GPChol dereased y 3.% and.%. The GPEtn/GPChol ratios signifiantly inreased in oth tissues, to 3.6±1.1 in the musle (P=., t-test), and to.7±.1 in the fat ody (P=.37, t-test). Changes were oserved also at the level of Relative aundane A GPEtns B GPChols m/z=7 8 amu m/z= amu 16:/18:-GPEtn m/z= amu 16:/18:1-GPEtn m/z= amu 17:/18:-GPEtn m/z= amu -GPEtn m/z= amu -GPEtn m/z= amu 18:/18:-GPEtn m/z= amu 18:/18:1-GPEtn Retention time (min) m/z=7 8 amu m/z=78 79 amu 16:/18:-GPChol m/z= amu 16:/18:1-GPChol m/z= amu -GPChol m/z= amu -GPChol m/z= amu 18:/18:-GPChol Fig. 3. HPLC separation of major moleular speies of (A) glyerophosphoethanolamines (GPEtns) and (B) glyerophosphoholines (GPChols) of P. apeterus thorai musle tissue. The upper most trae (in A and B) shows total GPEtn and GPChol frations and the lower traes show individual ion masses (moleules), as they were extrated using the MS ion trap. Note that a relative y axis are used in all hromatograms, where the highest peak is taken as % response. That is why the signal/noise is relatively low in the rare speies, e.g. 16:/18:1-GPChol. For quantifiation purposes, the areas under hromatographi peaks of individual moleular speies derived from a total hromatogram (upper traes) were ompared.

7 48 A. Tomala and others FA omposition in oth tissues; the UFA/SFA ratios signifiantly dereased (Tale 4). PCA analysis PCA identified two prinipal omponents (PC1 and PC), whih together aounted for 8.8% of the total variation seen among different treatments (field and laoratory data were analyzed in a single run). Fig. 8A presents the sores of the two prinipal omponents for individual treatments. In the twodimensional spae desried y PC1 and PC sores, vetors may y drawn, whih onnet an initial state with a state after the appliation of a treatment. Similar patterns in hanges of GPL omposition appear as similar orientations of the vetors. The length of eah vetor is proportionally related to the signifiane of the hange. The vetors displaying the autumnal alimatization in the field and the old alimation in the laoratory were relatively long and of similar orientation in oth tissues. The differenes etween the relevant sores of prinipal omponents were signifiant at =. (ANOVA followed y SNK test) (Fig. 8B,C). In ontrast, the vetors related to diapause progression and desiation (these were not drawn in Fig. 8A) were relatively short and the differenes etween the sores were mostly insignifiant. Disussion Complex nature of old alimation Inset old alimation is a omplex physiologial hange onsisting of many omponents (Lee, 1991; Danks, 1996; Renault et al., ; Sinlair et al., 3) of whih the memrane restruturing is just one. Hene, it is diffiult to estimate the effet of any single omponent in isolation. In P. Relative proportion (%) 3 A Otoer GPChols GPEtns Relative proportion (%) 3 A Otoer GPChols GPEtns 16:/18: 16:/18:1 17:/18: 18:/18: 18:/18:1 16:/18: 16:/18:1 17:/18: 18:/18: 18:/18:1 Change in relative proportion (%) 8 B Deemer :/18: 16:/18:1 17:/18: 18:/18: 18:/18:1 Change in relative proportion (%) 8 B Deemer :/18: 16:/18:1 17:/18: 18:/18: 18:/18:1 Fig. 4. Restruturing of glyerophospholipid omposition in thorai musle memranes of Pyrrhooris apterus during field alimatization etween 3 Otoer and 7 Deemer. (A) Mean (± s.d.; N=6 independent samples) relative proportion of phosphatidylethanolamine (GPEtn) and phosphatidylholine (GPChol) moleule (all speies make % in total) in Otoer. (B) The differene from the initial state at the end of the alimatization period. The differenes were statistially analyzed using unpaired twotailed t-tests (P<.; P<.1; P<.1). Fig.. Restruturing of glyerophospholipid omposition in fat ody tissue memranes of Pyrrhooris apterus during the field alimatization etween 3 Otoer and 7 Deemer. (A) Mean (± s.d.; N=6 independent samples) relative proportion of phosphatidylethanolamine (GPEtn) and phosphatidylholine (GPChol) moleule (all speies make % in total) in Otoer. (B) The differene from the initial state at the end of the alimatization period. The differenes were statistially analyzed using unpaired twotailed t-tests (P<.; P<.1; P<.1).

8 Cold alimation of memranes 49 Tale 3. Composition of fatty ayls in glyerophospholipids extrated from two tissues of the field-olleted adults of Pyrrhooris apterus Relative proportion (%) of the fatty ayl Fatty ayl In Otoer In Deemer Musle FA 16: 13.8±.6 16.±.6 FA 17: 1.4±.1 1.3±.1 FA 18:.±.8.±.9 FA 18:1 7.8±. 7.3±. FA 18: 66.8±.9 6.±.8 UFA/SFA.9±.1.6±.1 Fat ody FA 16: 14.1±1. 16.±.6 FA 17:.±..3±.4 FA 18: 1.3± ±.8 FA 18:1.7±.6.4±.7 FA 18: 6.8± ±.6 UFA/SFA.8±..6±.1 FA, fatty ayl; UFA, unsaturated FA; SFA, saturated FA; 16:, palmitoyl; 17:, heptadeanoyl; 18:, stearoyl; 18:1, oleyl; 18:, linoleyl. Eah value represents the mean ± s.d. of the relative proportion of the FA (or UFA/SFA ratio) alulated from phospholipid speies omposition. The differenes etween Otoer and Deemer means were statistially analyzed using unpaired two-tailed t-tests (P<.; P<.1; P<.1). apterus, depression of SCP temperature down to etween and C is neessary to extend the range of survival temperatures. This phenomenon was oserved repeatedly in the field (Kostál and Simek, ) (Tale 1) and in the laoratory old-alimation experiments (Hodková and Hodek, 1997; Kostál et al., 1) (Tale ). The low SCP alone, however, does not guarantee high levels of hill tolerane (Bale, 1987). Aout % of diapausing P. apterus adults showed SCP lower than C already prior to old alimation (oth in the field and in the laoratory, see the values of 7 perentiles in Tales 1 and ). The minimum SCP values in suh adults was C. Despite that, none of those adults survived longer than 1 day at C (Tales 1, ). This shows learly that additional adjustments are required to reah the maximum hill tolerane. Among them, aumulation of riitol and soritol play an important role (Kostál and Simek, ; Kostál et al., 1; Kostál et al., 4a). Both ompounds were also found in old-alimated P. apterus in relatively high onentrations in this study (Tales 1, ). Enhaning the apaity to maintain ion gradients aross ell memranes and epithelia at su-zero temperatures is another adjustment that proaly ontriutes to inreasing hill tolerane of old-alimated P. apterus (Kostál et al., 4). The potential ontriution of memrane restruturing to the inreasing hill tolerane of this inset was hypothesized first y Hodková et al. (Hodková et al., ). They desried the Relative proportion (%) Change in relative proportion (%) 3 A state GPChols GPEtns 4 B progression 16:/18: 16:/18:1 17:/18: 4 C D Cold-alimation :/18: 16:/18:1 17:/18: differenes in the GPL omposition of memranes etween laoratory, warm-alimated insets and those olleted in the field during Feruary [fig. 4 in Hodková et al. (Hodková et al., 18:/18: 18:/18: 18:/18:1 18:/18:1 Fig. 6. Restruturing of glyerophospholipid omposition in thorai musle memranes of Pyrrhooris apterus after various laoratory alimations. (A) state; (B) dipause progression; (C) desiation; (D) old alimation. The differenes were statistially analyzed using unpaired two-tailed t-tests (P<.; P<.1; P<.1).

9 41 A. Tomala and others Relative proportion (%) Change in relative proportion (%) 3 A state GPChols GPEtns 4 B progression 16:/18: 16:/18:1 17:/18: 4 C 6 D Cold-alimation :/18: 16:/18:1 17:/18: 18:/18: 18:/18: 18:/18:1 )]. We verified and further extended their results using more advaned analytial tehniques and omparing the responses in the field and variously treated laoratory animals. Autumnal alimatization and hanges in GPL omposition The inrease in the relative proportion of 1-palmitoyl-- linoleyl-sn-gpetn at the expense of 1,-dilinoleyl-sn-GPChol 18:/18:1 Fig. 7. Restruturing of glyerophospholipid omposition in fat ody tissue memranes of Pyrrhooris apterus after various laoratory alimations. (A) state; (B) dipause progression; (C) desiation; (D) old alimation. The differenes were statistially analyzed using unpaired two-tailed t-tests (P<.; P<.1; P<.1). was the most prominent speifi hange oserved in the wild adults of P. apterus during their autumnal alimatization in the field. It was one omponent of a more general trend, i.e. the inrease in the total proportion of GPEtn speies ounterated y the derease in the total proportion of GPChol speies (Fig. 3B, Fig. 4B), whih also an e expressed as an inrease in the GPEtn/GPChol ratio. Similar hanges were registered in two tissues, thorai musle and fat ody. It should e noted here that whereas the loomotory funtion of musle does not hange seasonally, the physiology of fat ody hanges profoundly. The fat ody is the prinipal tissue for intermediary metaolism in insets (Keeley, 198). Its major funtion in reprodutively ative females is the synthesis and release of vitellogeni proteins for yolk formation during ooyte maturation (Telfer, 3). At early phases of diapause, the fat ody synthesizes large amounts of energy reserves in the form of diapause proteins, triaylglyerols and glyogen. During the later phases of diapause, the syntheti ativity of the fat ody delines and the lipidi and glyogen reserves are slowly utilized (Tauer et al., 1986). Upon stimulation y low temperature, the fat ody glyogen eomes the main soure for the rapid iosynthesis of polyol ryoprotetants (Storey and Storey, 1991). The extensive seasonal restruturing of GPL omposition in P. apterus ourred without major hanges in the proportions of the FAs. A moderate inrease (y.4%) in the proportion of palmitoyl (16:) was found in oth tissues (Tale 3). The most ommon response of etotherm memranes to old is to inrease the proportion of unsaturated FAs and/or inrease the numer of doule onds per FA. This response was not seen in P. apterus. In fat, autumnal alimatization led to a slight derease in the UFA/SFA ratio in oth tissues (Tale 3). Our FA data, however, were not otained y diret analysis of FAs ut were derived from original analytial data of GPL speies. Nevertheless, diret GC/MS analysis of FAs in P. apterus [performed y Hodková et al. (Hodková et al., 1999)] onfirmed a positive orrelation etween the unsaturation ratio and amient temperature, and a negative orrelation etween the proportion of palmitoyl and temperature. In the field, various fators triggering/stimulating GPL restruturing may not e easily distinguished. Our laoratory experiments allowed us to separate three potential fators: low temperatures, desiation, and endogenous fators related to diapause progression. Whereas the low temperatures stimulated signifiant hanges (Fig. D, Fig. 6D), the later two fators appeared to have relatively small effets on GPL omposition in P. apterus memranes. Triggering y low temperatures The hanges in GPL omposition triggered y low temperatures in the laoratory were similar to the alimatization hanges oserved under the field situation. The GPEtn/GPChol ratio inreased, as a result of the inreases in 16:/18:-GPEtn and 18:/18:-GPEtn ountered y the derease in -GPChol. Results of multivariate statistial analysis (PCA) further supported the overall

10 Cold alimation of memranes 4111 similarity etween the restruturings seen in the field and laoratory and in oth tissues (Fig. 8). This was so despite the fat that the initial states widely differed etween the two situations and etween the two tissues as well. Suh uniformity in the response suggests a ommon adaptive value of the underlying GPL hanges in relation to old. The ratio of GPEtn/GPChol generally tends to e higher in old- than in warm-alimated etotherms (Hazel, 1989; Hazel, 199; Hazel, 1997). Moleules of the two lipid lasses differ in their geometries: GPChols assume a ylindrial shape and pak effiiently in a lamellar gel phase; GPEtns adopt a more onial form, whih is readily aommodated in the reverse hexagonal phase. Consequently, less effiient paking of GPEtns in the lamellar phase disrupts the organization of the memrane and thus ounters the effets of low temperatures, whih tend to organize the memrane in a gel phase (Hazel, 1989). The inreasing GPEtn/GPChol ratio in a memrane may thus help to prevent the formation of a gel phase in memranes at low amient temperatures. In our earlier work (Slahta et al., a), we deteted a slight inrease in the GPEtn/GPChol ratio in response to low temperatures in the non-diapausing adults of P. apterus. It inreased from 1. to 1.1 (NS, t-test) in the musles, and from.7 to 1. (P=.3, t-test) in the fat ody. Non-diapausing adults were not ale, however, to aomplish the other old-alimation adjustments seen in diapausing adults (SCP depression, polyol aumulation and ion gradients regulation) and no, or only a weak, inrease in their hill tolerane was oserved (Slahta et al., a). It is more diffiult to searh for an adaptive explanation for the oserved hanges in relative proportions of speifi moleular speies, suh as the 16:/18:-GPEtn. The range of temperatures at whih memranes omposed of GPEtns remain A PC sore (18.9% of variation) Musle Cold Otoer Deemer Otoer Deemer Fat ody Cold PC sore B a Fat ody Otoer Deemer 3 PC1 a Musle PC1 sore (64.4% of variation) Cold Otoer Deemer Cold C a Fat ody a a Otoer Deemer PC Cold a,, Musle Otoer Deemer d Cold Fig. 8. Results of multivariate prinipal omponent analysis of the differenes etween memrane glyerophospholipid ompositions in thorai musles and fat ody tissues of Pyrrhooris apterus sampled in the field (3 Otoer and 7 Deemer) or variously alimated in the laoratory (initial state, diapause progression, desiation, old alimation). (A) The entres of elipses represent the means, and the orders the standard errors, of the sores from the first and seond prinipal omponent. The dashed line separates the data for musles and fat-ody tissues. (B,C) The differenes among the sores of PC1 and PC, respetively, were tested using one-way ANOVA followed y a Student Newman Keuls test (different letters indiate statistially different values).

11 411 A. Tomala and others Tale 4. Composition of fatty ayls in glyerophospholipids extrated from two tissues of the laoratory-reared adults of Pyrrhooris apterus, and the effets of diapause progression, desiation and old alimation Relative proportion (%) of the fatty ayl state Cold Fatty ayl (day ) progression ( months) (14 days) alimation ( months) Musle FA 16: 13.3±.9 14.± ± ±. FA 17: 1.4±. 1.3±.3 1.4±. 1.±.1 FA 18: 13.4± ± ±1..4±1.8 FA 18:1 9.±.8 9.±. 9.±.6 8.±1.3 FA 18: 6.3± ± ±. 6.±1.9 UFA/SFA.6±.1.7±..4±..±.1 Fat ody FA 16: 13.9± ±.8 1.± ±.6 FA 17:.1±.1.6±..4±.7.6±.4 FA 18: 1.1± ± ± ±.6 FA 18:1 1.9± ± ± ±.9 FA 18: 61.1±. 9.3±1. 9.4± ±1. UFA/SFA.8±.1.8±.1.8±.1.6±.1 Eah value represents the mean ± s.d. of the relative proportion of the FA (or UFA/SFA ratio) alulated from phospholipid speies omposition. The differenes etween the initial state and the treatments were statistially analyzed using unpaired two-tailed t-tests (P<.; P<.1; P<.1). fluid, i.e. T m T h (where T m is a temperature of transition from lamellar gel to liquid-rystalline phase and T h is a temperature of transition from liquid-rystalline to reverse hexagonal phase) markedly inreases with a derease in ayl hain length (Lewis et al., 1989). Hodková et al. (Hodková et al., ) suggested that a relative inrease of 16: FA and its speifi pairing with 18: FA in the GPEtn moleule may help to maintain the amient temperature within a suitale interval aove T m yet elow T h, and protet memranes from unregulated phase transitions aused y either a drop of amient temperature or partial dehydration, or oth ourring at the same time, as is normal in overwintering insets. Generally, the phase ehaviour, fluidity and other properties of memranes are finely tuned to serve various purposes in different environmental situations. Changing the lipidi omposition is the main tool of suh tuning. Some hanges may represent just a side effet without any expliit adaptive value. However, inreasing the relative proportion of 16:/18:- GPEtn, as a speifi response to old, might e of some adaptive value as it was found in two tissues with different physiologial funtions, and it was oserved also in the fly, Chymomyza ostata (Kostál et al., 3), and water strider (M. Hodková, unpulished results), whih are eologially and phylogenetially distant to P. apterus. Triggering y desiation It has een proposed that some of the physiologial adaptations to old might e derived from more anestral adaptations to desiation (Ring and Danks, 1994; Blok, 1996; Danks, ; Williams et al., 4). Populations of P. apterus proaly olonized entral Europe from Asia Minor and the Near East relatively reently, during the limate warming after the end of the last glaial period. The anestral haitats were prone to drying, whih ould drive the evolution of physiologial adaptations to desiation in this speies. Moreover, P. apterus adults typially lose up to one third of ody water during old alimation and overwintering (Kostál and Simek, ; Kostál et al., 4) (Tales 1, ). In this study, we attempted to test whether desiation triggers some hanges in GPL omposition. In the ollemolan, Folsomia andida, a mild desiation stress was suffiient to eliit the restruturing of memrane GPLs (Bayley et al., 1; Holmstrup et al., ). In addition to alteration of memrane lipids, Folsomia andida responded to desiating onditions y aumulating gluose and myo-inositol, whih led to the inrease in osmolality, and susequently influened the rates of water uptake/loss (Bayley and Holmstrup, 1999). Our experiments showed that the loss of one half of ody water, assoiated with douling the haemolymph osmolality, indued relatively small hanges in GPL omposition in P. apterus and that the overall pattern of these hanges differed from the hanges indued y low temperatures (Fig. 7). For example, a derease in the proportion of 16:/18:-GPEtn was noted in response to desiation whereas an inrease was deteted as a result of old alimation (see aove). No, or negligile, aumulation of polyhydri solutes was oserved in response to desiation in P. apterus (Tale ). Triggering y endogenous fators related to diapause The preparation for winter survival starts long efore the autumnal deline of environmental temperatures in P. apterus (Kostál and Simek, ), and in many other insets as well

12 Cold alimation of memranes 4113 (Danks, 1987; Kostál, 6). Hodková et al. (Hodková et al., ) found that the relative proportion of 16:/18:-GPEtn was higher in short-day reared (diapausing) than in long-day reared adults of P. apterus (non-diapausing, reproduing) that were maintained in the laoratory at 6 C. Moreover, when non-diapausing females of P. apterus had the orpus allatum surgially removed (whih prevents juvenile hormone prodution and auses entry into a diapause-like state), the GPL profiles in their musle and fat ody tissues hanged in a similar manner to those of females in whih diapause was indued photoperiodially (Hodková et al., ). Similar results were otained with larvae of the fly Chymomyza ostata, where relative proportions of 16:/18:-GPEtn in total GPLs extrated from the musle and fat ody tissues signifiantly inreased in response to oth low temperature stimulus and the transition from diret development to diapause, i.e. without any hange in temperature (Kostál et al., 3). Suh results suggest that the seasonal restruturing of GPL omposition is photoperiodially ativated already during the entrane into diapause, whih typially happens (long) efore the seasonal deline in temperatures, and that the hanges are hormonally mediated. By ontrast, the hanges in GPL omposition during the diapause progression, as desried in this paper, were relatively small. Moreover, the orientations of the vetors of diapause-related hanges (Fig. 8A) were opposite in the musle and fat ody tissues. Suh a lear differene in the vetor orientations might reflet speifi (hanges of) physiologial roles in two different tissues during diapause, whih may require synthesizing/ maintaining speifi GPL speies. In this light, relatively deep and uniform restruturing of GPL omposition of oth tissues in response to old implies an adaptive mehanism assisting in survival at low temperatures. The study was supported y the Czeh Siene Foundation grant no. 6/3/99. Referenes Allakhverdiev, S. I., Nishiyama, Y., Suzuki, I., Tasaka, Y. and Murata, N. (1999). Geneti engineering of the unsaturation of fatty aids in memrane lipids alters the tolerane of Synehoystis to salt stress. Pro. Natl. Aad. Si. USA 96, Bale, J. S. (1987). Inset old hardiness: freezing and superooling an eophysiologial perspetive. J. Inset Physiol. 1, Bayley, M. and Holmstrup, M. (1999). Water vapor ansorption in arthropods y aumulation of myoinositol and gluose. Siene 8, Bayley, M., Petersen, S. 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